Project summary Influenza A virus (IAV) is a highly transmissible respiratory pathogen and presents a continued threat to global health, with considerable economic and social impact. IAV comprises a plethora of strains with different virulence determinants that contribute to influenza pathogenesis. Several determinants in non-structural protein 1 (NS1) of high pathogenic IAV strains have been found to subvert host defense and increase virulence. However, the NS1 of 2009 pandemic IAV (2009 IAV) lacks the known determinants. To date, little is known about the mechanisms underlying how NS1 of 2009 IAV interacts with the host and how the interactions control host defense and viral pathogenesis. Answering these questions will reveal new host defense mechanisms and new NS1 virulence factors. To discover the new virulence determinant, we recently systematically analyzed NS1 protein complexes and found that the host factor, zinc finger C3H1-type containing protein (ZFC3H1), specifically interacted with NS1 of 2009 IAV. ZFC3H1 is known to form the poly(A) tail exosome targeting complex (PAXT) with the ATP- dependent RNA helicase MTR4 and the nuclear poly(A)-binding protein (PABPN1). PAXT directs the RNA exosome to degrade mRNA, but whether the PAXT complex limits viral infection is unknown. Our pilot experiments have revealed that ZFC3H1 mediates RNA exosome to promote viral RNA degradation, thereby limiting influenza replication. Based on the existing literature and our preliminary data, we propose the central hypothesis that ZFC3H1 inhibits influenza virus infection and that this inhibition is counteracted by the NS1 encoded by the 2009 IAV strains. Aim 1 will determine the mechanisms by which ZFC3H1 limits IAV infection. Aim 2 will determine the mechanisms by which the NS1 of 2009 IAV impairs ZFC3H1-mediated defense. Aim 3 will determine the ZFC3H1-IAV interaction in animal models. Completion of this proposal will reveal a new host defense mechanism by which ZFC3H1 enhances the RNA decay machinery through PAXT-mediated viral mRNA degradation and will provide insight into an exquisite viral strategy for the evasion of host defense surveillance through engagement between NS1 and ZFC3H1. Our study will not only help develop effective anti-viral therapeutics but will also deepen our understanding of host defense against influenza.